The long-term goal of this research is to elucidate the means by which Epstein-Barr virus (EBV) induces and maintains proliferation of the human B-lymphocytes that it infects. EBV causes or contributes causally to several human diseases of B-lymphocytes including infectious mononucleosis, Burkitt's lymphoma, and lymphomas in immunocompromised patients. It is likely that it does so by causing indefinite proliferation of the B-lymphocytes it infects. It is therefore particularly desirable to understand the process of immortalization of cells by EBV in order to comprehend and eventually to ameliorate the lymphoid diseases associated with EBV. At least three viral proteins, EB nuclear antigens 1 and 2 (EBNA 1 and 2) and the latent membrane protein (LMP) appear to be involved in immortalization. these proteins will be studied to identify their contributions to the process of immortalization. These proteins will be studied to identify their contributions to the process of immortalization. EBNA-1 mediates replication of EBV plasmid DNA and can transactivate transcription. It has been purified so that its intrinsic activities can be identified and analyzed. In particular, its ability to link two DNA molecules to which it binds will be dissected mechanistically in vitro; post-translational modifications that might control its function will be characterized after its isolation from synchronized cells; and cellular proteins with which it interacts to affect transcription will be identified by methods that use crosslinking and by functional tests that employ transcriptional assays in vitro. EBNA-2 affects the transcription of viral and cellular genes variably in different clones of B-lymphoblasts. In order to identify genetic elements affected by EBNA-2 in EBV-immortalized cells, B-lymphoblasts will be engineered to express a chimera of EBNA-2 and a steroid receptor such that the function of EBNA-2 will be dependent upon addition of the steroid. Cis-acting sites that affect viral proteins that are dependent upon function EBNA-2 will be identified and analyzed to find those cellular binding proteins with which EBNA-2 acts to mediate its functions. LMP is likely to contribute to immortalization of cells by EBV by affecting some facet of cellular signaling. An assay in B- lymphoblasts that responds rapidly to LMP will be developed and used to identify pharmacologically specific signaling pathways with which LMP is likely to interact. Experiments that use crosslinking will also be employed to identify cellular proteins that associate with LMP in the expectation that their identification will help to elucidate functions of LMP. Finally the requirement for the continued expression of each of these viral proteins to maintain proliferation of EBV-infected cells will be tested genetically.